Summary The use of opioids, such as morphine, as analgesics for the treatment of chronic pain is a common clinical practice. However, a major disadvantage of long-term opioid treatment is the development of opioid-induced bowel dysfunction, which commonly manifests with severe constipation, occurring in more than 40% of the patients. Analgesia and constipation are mediated by the interaction of opioids with ? opioid receptors (?ORs). However, constipation is mediated by ?ORs in enteric neurons, whereas analgesia is mediated by ?ORs in the central nervous system (CNS). Furthermore, analgesia undergoes tolerance with long-term treatment, whereas constipation does not subside over time and worsens with the increase of analgesic doses required to control pain. Activation of ?ORs initiates a cascade of events including endocytosis, desensitization and resensitization. Endocytosis and desensitization are key components in reducing signaling, and long-term treatment induces intracellular changes leading to adaptations in downstream signaling in heterologous cells and CNS neurons. In contrast, ?OR signaling events triggered by long-term opioids in enteric neurons are largely unknown and our initial studies indicate they differ from CNS neurons. The overarching hypothesis of this proposal is that long-term activation of ?OR in enteric neurons leads to signaling events mediating the development of constipation, which is resistant to tolerance. This is in contrast to induction of tolerance in the CNS. These studies will use rats and mice, and in vivo and in vitro approaches with cultured enteric neurons, patch clamp recording, lentiviral neuronal transfection to block trafficking proteins, and mice with deletion of signaling molecules or opioid receptors. Specific Aim 1 will test the hypothesis that downstream signaling events in enteric neurons following long-term opioids are ligand dependent and tissue specific. cAMP, MAPK phosphorylation and Ca2+ channel currents will be evaluated as measures of receptor-effector coupling. Specific Aim 2 will identify the relationship between ?OR internalization and desensitization in response to morphine and internalizing agonists, determine the importance of ?OR internalization in downstream signaling and the specific roles of trafficking proteins and kinases involved in ?OR regulation. Specific Aim 3 will identify the relationship between ?OR signaling events and GI and colonic transit delay induced by long-term ?OR activation and determine whether disruption of ?OR signaling affects opioid-induced GI motility impairment. Understanding the mechanisms underlying the development and persistence of constipation will provide better targets for its prevention and treatment. This proposal will have high impact in improving the quality of life of patients experiencing the burden of severe constipation.